I. Research Plan Thrombolytic therapy remains suboptimal because of difficulties with bleeding, incomplete lysis and rethrombosis. previous attempts to improve the safety and efficacy of this therapy have all by ignored alpha2-antiplasmin (alpha2AP), the chief inhibitor of clot lysis. In this application, we propose to study the functions of alpha2AP in fibrinolysis and to use the understanding acquired to develop and test a safer, more effective thrombolytic strategy. To study alpha2AP in plasma we have developed a highly specific monoclonal antibody that inhibits soluble and fibrin-crosslinked alpha2AP. Preliminary work with this antibody has confirmed the pivotal role played by alpha2AP in retarding fibrinolysis. By inhibiting alpha2AP this antibody is synergistic with tissue plasminogen activator (t-PA) in clot lysis; at the same time, it increases the specificity of t-PA for fibrinolysis. Using this antibody and other techniques, we plan to better define the special roles played by fibrin-crosslinked, platelet-derived, and other forms of alpha2AP on fibrinolysis. This information will be exploited to obtain a more specific inhibitory antibody which can act as a clot-specific, thrombolytic agent. Our long-term goal is to design a recombinant, hybrid thrombolytic agent consisting of this inhibitory antibody coupled to a plasminogen activator. II. Candidate Training In addition to meeting scientific objectives, this application has been formulated to achieve the research training goals of the applicant. The work described in the initial """"""""Specific Aims"""""""" of the proposal is designed to further expand the candidate's training in techniques of protein chemistry and immunology. Later phases of the proposal are designed to provide the candidate's with experience in testing hypotheses in an in vivo experimental system. Finally, the last phase is design to provide training in the techniques of cloning, as well as the design, expression and characterization of new, recombinant molecules. III. Research Environment The work outlined in this application will be conducted in the Laboratory of Cellular and Molecular Research at Massachusetts General Hospital. This laboratory has had particular success in the scientific training of physicians. The laboratory currently has 19 postdoctoral fellows and 9 faculty engaged in a broad range of basic cardiac research. Regular laboratory meetings and postdoctoral seminars are a regular source for critical scientific evaluation and exposure to new techniques. There is also the broader educational interaction afforded by the Harvard Combined Seminar Series in Atherosclerosis and Vascular Biology. Consistent with the initial scientific training goals of the applicant, the sponsor will be DR. Gary R. Matsueda, a biochemist with expertise in fibrinogen and fibrinolytic research, peptide synthesis and the elicitation of highly specific monoclonal antibodies. The co-sponsor, Dr. Thomas Quertermous is a molecular biologist with unique experience in the design and expression of recombinant, hybrid fibrinolytic agents. Dr. Matsueda and Quertermous have successfully collaborated with each other in achieving scientific goals as well as the training of young scientists.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL002348-03
Application #
3082703
Study Section
Research Manpower Review Committee (MR)
Project Start
1989-07-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Reed, G L; Houng, A K; Liu, L et al. (1999) A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator. Proc Natl Acad Sci U S A 96:8879-83
Reed, G L; Houng, A K; Bianchi, C (1998) Comparative biochemical and ultrastructural studies of P-selectin in rabbit platelets. Comp Biochem Physiol B Biochem Mol Biol 119:729-38
Parhami-Seren, B; Keel, T; Reed, G L (1997) Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage. J Mol Biol 271:333-41
Butte, A N; Houng, A K; Jang, I K et al. (1997) Alpha 2-antiplasmin causes thrombi to resist fibrinolysis induced by tissue plasminogen activator in experimental pulmonary embolism. Circulation 95:1886-91
Reed, G L (1997) Functional characterization of monoclonal antibody inhibitors of alpha 2-antiplasmin that accelerate fibrinolysis in different animal plasmas. Hybridoma 16:281-6
Houng, A K; Maggini, L; Clement, C Y et al. (1997) Identification and structure of activated-platelet protein-1, a protein with RNA-binding domain motifs that is expressed by activated platelets. Eur J Biochem 243:209-18
Parhami-Seren, B; Keel, T; Reed, G L (1996) Structural characterization of immunodominant regions of streptokinase recognized by murine monoclonal antibodies. Hybridoma 15:169-76
Lin, L F; Oeun, S; Houng, A et al. (1996) Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex. Biochemistry 35:16879-85
Parhami-Seren, B; Lynch, M; White, H D et al. (1995) Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy. Mol Immunol 32:717-24
Reed, G L; Lukacova, D (1995) Generation and mechanism of action of a potent inhibitor of factor XIII function. Thromb Haemost 74:680-5

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